Encapsulated intestinal midgut endoderm cells

ABSTRACT

Cell populations of intestinal midgut endoderm cells and methods of generating the cells expressing markers characteristic of intestinal endoderm lineage are disclosed. Methods of treating conditions such as diabetes are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/478,881, filed Apr. 4, 2017, which claims the benefit of U.S.Provisional Application No. 62/322,636, filed Apr. 14, 2016. Theabove-listed applications are herein incorporated by reference in theirentirety.

FIELD OF INVENTION

The invention relates to the field of cell-based therapy for conditionssuch as diabetes. In particular, the invention relates to celldifferentiation, including directing differentiation of humanpluripotent stem cells to generate a population of intestinal midgutendoderm cells. The invention provides cells or a cell population andmethods of producing the cells that express markers characteristic ofintestinal midgut endoderm.

BACKGROUND

Advances in the knowledge of incretin hormone mechanism of actioncoupled with advancements in the understanding of intestinaldifferentiation, both at the stem cell and endocrine cell stages, haveled to interest in developing sources of incretin hormone producingcells, appropriate for engraftment. One approach is the generation offunctional enteroendocrine L- or K-cells from pluripotent stem cells,such as human embryonic stem cells (“hESC”) or induced pluripotent stemcells (“iPS”).

The production/secretion of glucagon-like peptide 1 (GLP-1) fromintestinal L-cells or glucose-dependent insulinotropic polypeptide (GIP)from intestinal K-cells has beneficial effects for the treatment ofdiabetes mellitus. Incretin hormones have systemic effects beneficialfor the treatment of diabetes mellitus (Type 1 and Type 2) (Unger, J.,Curr Diab Rep., 2013; 13(5):663-668). Benefits may include augmentationof many aspects of beta (β) cell function and number, suppression ofglucagon secretion, increases in the insulin sensitivity of peripheralmetabolic tissues, reduction of hepatic gluconeogenesis, and reductionof appetite. Two classes of incretin-based therapeutic agents have beenidentified for the treatment of diabetes mellitus (GLP-1 receptoragonists and dipeptidyl peptidase 4 (DPP-4) inhibitors). However, thereis currently no incretin-based cell therapy option that would encompassan endogenous and cellular barometer for improved and efficientGLP1-based diabetes treatment. Furthermore, current incretin-basedtherapies are not regulated by circulating blood glucose levels and thusprovide non-physiologically regulated GLP production.

In vertebrate embryonic development, a pluripotent cell gives rise to agroup of cells comprising three germ layers (ectoderm, mesoderm, andendoderm) in a process known as gastrulation. The mesenchyme tissue isderived from the mesoderm, and is marked by the genes heart and neuralcrest derivatives expressed 1 (HAND1), and forkhead box F1 (FOXF1),among others. Tissues such as, thyroid, thymus, pancreas, gut and liver,will develop from the endoderm, via an intermediate stage. Theintermediate stage in this process is the formation of the definitiveendoderm. By the end of gastrulation, the endoderm is partitioned intoanterior-posterior domains that can be recognized by the expression of apanel of factors that uniquely mark anterior foregut, posterior foregut,midgut, and hindgut regions of the endoderm.

The level of expression of specific transcription factors (“TFs”) may beused to designate the identity of a tissue, as described inGrapin-Botten et al., Trends Genet, 2000; 16(3):124-130. FOXA2 marks theentire endoderm along the anterior-posterior axis. During transformationof the definitive endoderm into a primitive gut tube, the gut tubebecomes regionalized into broad domains that can be observed at themolecular level by restricted gene expression patterns. The anteriorforegut is marked broadly by the high expression of SOX2, andencompasses organ domains such as the thyroid, lung, and esophagus. Themidgut (includes the duodenum, ileum, jejunum) and hindgut (includes thecolon) are marked by high expression of caudal type homeobox 2 (CDX2).The SOX2-CDX2 boundary occurs within the posterior foregut, within whichadditional TFs mark specific organ domains. The regionalized pancreasdomain within the posterior foregut shows a very high expression of PDX1and very low expression of CDX2 and SOX2. PTF1A is highly expressed inpancreatic tissue. Low PDX1 expression, together with high CDX2expression marks the duodenum domain. The intestinal endoderm ispatterned by specific homeobox (HOX) genes. For example, HOXC5 ispreferentially expressed in midgut endoderm cells. In addition, theexpression of HOXA13 and HOXD13 are restricted to hindgut endodermcells. The ALB gene, or albumin 1 protein, marks the earliest liverprogenitors in the posterior foregut endoderm (Zaret et al., Curr TopDev Biol, 2016; 117:647-669).

Strides have been made in improving protocols to generate intestinalendoderm cells from human pluripotent stem cells. For example, thefollowing publications (Spence et al., Nature, 2011; 470(7332):105-109;Watson et al., Nature Medicine, 2014; 20(11):1310-1314; and Kauffman etal., Front Pharmacol, 2013; 4(79):1-18) outline differentiationprotocols using either fibroblast growth factor (FGF)-4, Wingless-typeMMTV integration site family, member 3A (WNT3A), Chiron 99021, orretinoic acid (RA) and FGF7 starting at the definitive endoderm stage,that generate mid-/hindgut spheroids, containing not only a CDX2⁺/FOXA2⁺endodermal population, but also a significant mesenchymal CDX2⁺ cellpopulation. The process of differentiating enteroendocrine cells fromthese hESC-derived mid-/hindgut spheroids is very inefficient, requiringa long time period, and is directed non-discriminately towards thegeneration of all intestinal cell types of the intervillus and villusregions. A need still exists for technology to generate intestinalmidgut endoderm cells, without substantial contaminating mesenchyme, soas to be able to produce with high efficiency intestinal enteroendocrinecells for cell therapeutics.

SUMMARY OF THE INVENTION

As embodied and fully described, the invention provides cells, cellpopulations and methods of generating the cells by differentiating humanpluripotent stem cells. In particular, the invention features methods ofdirected differentiation of human pluripotent stem cells, to generateintestinal midgut endoderm cells, more particularly an endodermalmonolayer of intestinal midgut endoderm cells.

One aspect of the invention is a method of producing a population ofintestinal midgut endoderm cells comprising culturing human pluripotentstem cells in culture media. In embodiments, the method comprisesinducing differentiation of human pluripotent stem cells to intestinalmidgut endoderm cells. In some embodiments, a population of intestinalmidgut endoderm cells is produced. In some embodiments, a population ofsubstantially intestinal midgut endoderm cells is produced. Inembodiments of the invention, the intestinal midgut endoderm cells formand are stable as a monolayer in culture. In embodiments, greater than50% of the differentiated cells express markers characteristic ofintestinal midgut endoderm, preferably greater than 60% of thedifferentiated cells express markers characteristic of intestinal midgutendoderm, more preferably greater than 70%, greater than 80%, andgreater than 90% express markers characteristic of intestinal midgutendoderm. In embodiments, differentiated cells express markerscharacteristic of intestinal midgut endoderm are intestinal midgutendoderm cells. In embodiments, the intestinal midgut endoderm cellsexpress CDX2 and FOXA2. In all embodiments, the intestinal midgutendoderm cells express transcription factors selected from SOX9, PDX1,KLF5 and HOXC5. In embodiments, the intestinal midgut endoderm cells donot express transcription factors selected from SOX2, ALB, PTF1A, HOXA13and LGR5.

In embodiments of the invention, human pluripotent stem cells aredifferentiated to intestinal midgut endoderm cells by steps including:a) culturing the human pluripotent stem cells in a first culture mediacontaining GDF-8 and a GSK3β inhibitor, such as MCX compound, to inducedifferentiation into definitive endoderm cells; b) culturing thedefinitive endoderm cells in a second culture media containing ascorbicacid and FGF7 to induce differentiation into primitive gut tube cells;and c) culturing the primitive gut tube cells in a third culture mediacontaining retinoic acid and BMP2 or BMP4 in acidic conditions to inducedifferentiation into intestinal midgut endoderm cells. In particularembodiments, acidic conditions is culture with BLAR medium. The pH ofthe acidic culture can range from 6.8 to 7.2. In the embodiments of theinvention, the intestinal midgut endoderm cells form a monolayer inculture. In embodiments, the monolayer of intestinal midgut endodermcells is maintained in culture.

Another embodiment of the invention is a method of treating a patientsuffering from or at risk of developing diabetes comprisingdifferentiating human pluripotent stem cells to intestinal midgutendoderm cells, and administering the differentiated intestinal midgutendoderm cells in a patient with diabetes. In embodiments, diabetes isType 1 or Type 2. In embodiments, administering the cells may be viaimplantation, injection or otherwise administration directly orindirectly to the site of treatment. In some embodiments, the intestinalmidgut endoderm cells are implanted in the body, such as in subcutaneousspace, omentum, liver, kidney, etc. Further embodiments encompassencapsulated delivery of the cells including encapsulation of macro- ormicro-encapsulation devices.

A further embodiments of the invention is a method of producingintestinal midgut endoderm cells comprising inducing differentiation ofdefinitive endoderm cells in culture to primitive gut tube cells. Inembodiments, the definitive endoderm cells are cultured in culture mediacontaining ascorbic acid and FGF7. In further embodiments, the primitivegut tube cells are cultured in culture media containing retinoic acidand BMP2 or BMP4. The primitive gut tube cells are differentiated tointestinal midgut endoderm cells. In some embodiments, primitive guttube cells are differentiated to intestinal midgut endoderm cells inacidic conditions (acidic culture media). In particular embodiments,acidic conditions is culture with BLAR medium. The pH of the acidicculture can range from 6.8 to 7.2. In the embodiments, the intestinalmidgut endoderm cells form and maintain a monolayer in culture.

In each of the embodiments discussed above, human pluripotent stem cellsare human embryonic stem cells or induced pluripotent stem cells. Ineach of the embodiments above, the intestinal midgut endoderm cellsexpress CDX2 and FOXA2. In all embodiments, the intestinal midgutendoderm cells express transcription factors selected from SOX9, PDX1,KLF5 and HOXC5. In the embodiments, the intestinal midgut endoderm cellsdo not express transcription factors selected from SOX2, ALB, PTF1A,HOXA13 and LGR5. In the embodiments above, the intestinal midgutendoderm cells express CDX2, FOXA2, SOX9, PDX1, KLF5 and HOXC5. In theembodiments above, the intestinal midgut endoderm cells do not expressSOX2, ALB, PTF1A, HOXA13 and LGR5. In embodiments, greater than 50% ofthe differentiated cells express markers characteristic of intestinalmidgut endoderm, preferably greater than 60% of the differentiated cellsexpress markers characteristic of intestinal midgut endoderm, morepreferably greater than 70%, greater than 80%, and greater than 90%express markers characteristic of intestinal midgut endoderm. Inembodiments, differentiated cells express markers characteristic ofintestinal midgut endoderm are intestinal midgut endoderm cells. Inembodiments, the intestinal midgut endoderm cells express CDX2 andFOXA2. In embodiments, the intestinal midgut endoderm cells expresstranscription factors selected from SOX9, PDX1, KLF5 and HOXC5. Inembodiments, the intestinal midgut endoderm cells do not expresstranscription factors selected from SOX2, ALB, PTF1A, HOXA13 and LGR5.In the embodiments, intestinal midgut endoderm cells do not expressHAND1.

In the embodiments discussed above, the population of intestinal midgutendoderm cells is substantially intestinal midgut endoderm cells. Insome embodiments, the population of intestinal midgut endoderm cellscomprises greater than 70% intestinal midgut endoderm cells, preferablygreater than 80%, greater than 90%, and greater than 95% of intestinalmidgut endoderm cells. In some embodiments, the population of intestinalmidgut endoderm cells comprises less than 20% mesenchymal cells,preferably less than 15%, more preferably less than 10%, less than 5%,less than 2%, less than 1%, less than 0.5%. In embodiments, intestinalmidgut endoderm cells lack expression of HAND1.

In some embodiments of the invention described above, differentiation isinduced in vitro. In other embodiments, intestinal midgut endoderm cellsdifferentiate further in vivo. Another embodiment relates to theintestinal midgut endoderm cells further differentiating intoenteroendocrine cells in vivo. The enteroendocrine cells express orsecrete incretin hormones. In embodiments, the incretin hormones areGLP1 and GIP.

In a further embodiment, intestinal midgut endoderm cells serve asstarting material for the identification of small molecules that promoteat high efficiency the in vitro differentiation of intestinal midgutendoderm cells into, first, enteroendocrine precursors, and ultimately,incretin expressing or secreting enteroendocrine cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict a differentiation method for intestinal midgutendoderm cells. FIG. 1A is a summary of the differentiation method,including medium components, growth factors and small molecules added toeach stage, and key stage-specific markers of the differentiatingintestinal midgut endoderm cells (FOXA2, forkhead box A2; CDX2, caudaltype homeobox 2; KLF5, Kruppel-like factor 5; SOX9, SRY (sex determiningregion Y)-box 9; PDX1, pancreatic and duodenal homeobox 1; LO, lowexpression and protein presence). Compared to the neutral pH noted atS2D2 (7.35±0.04), cells were exposed to slightly acidic conditions inBLAR medium (used interchangeably with “BLAR acidic medium”), duringstage 3 (pH; S3D1, 6.98±0.05; S3D2, 7.02±0.04; S3D5, 7.18±0.03) (FIG.1B), and as a result of lower sodium bicarbonate levels in BLAR medium.FIG. 1C depicts representative phase-contrast images of a S3D5 monolayer(left), and human epithelial colon adenocarcinoma cell line (“Caco-2”)(right), which was used as a benchmark for characterization ofdifferentiation. A uniform morphology at S3D5 was consistently observed.Characterization of cell number using the Nucleocounter® NC-100(Chemometec, Alleroed, Denmark , Catalog No. 900-004) shows that onehESC differentiated into 4.56±2.60 S3D5 intestinal midgut endoderm cells(FIG. 1D).

FIGS. 2A-2D demonstrate the differentiation method, utilizing Bonemorphogenic protein-4 (BMP4), generates intestinal midgut endoderm cellsin monolayer, comprising both CDX2 and FOXA2 on transcript and proteinlevels. FIG. 2A (bottom) shows that 90.0±5.85 percent of S3D5 cells wereco-present for both CDX2 and FOXA2 protein, similar to percentage seenin Caco-2 cells (86.0±6.67). Conversely, definitive endoderm (DE—S1D3)cells were devoid of CDX2 and FOXA2 co-presence (2.3±1.2). Geneexpression analysis shows that CDX2 was induced (FIG. 2B), and FOXA2maintained (FIG. 2C) during Stage 3. FIG. 2D shows that the induction ofCDX2 protein levels and CDX2/FOXA2 protein co-presence after theestablishment of the FOXA2-positive primitive gut endoderm stage, S2D2(FIG. 2D-i), progressively increased through S3D2 (FIG. 2D-ii), and atS3D5 (FIG. 2D-iii) reached similar levels as seen in Caco-2 cells (FIG.2D-iv). CDX2 protein is depicted on the bottom row, and FOXA2 protein isdepicted on the top row. Each image was taken using the same parametersto allow for quantitative analysis. Protein expression was assessed byFACS; gene expression was assessed by qPCR.

FIGS. 3A-3Q demonstrate the induction by S3D5 of transcript and proteinlevels of additional transcription factors (TF) that constitute robustintestinal midgut endoderm induction; proper intestinal midgut endodermwas achieved. In addition to CDX2 and FOXA2 co-expression, S3D5 cellsalso exhibited co-expression of SOX9, PDX1, KLF5, HOXC5 (homeobox C5),but not SOX2 (SRY-box 2), ALB (albumin), PTF1A (pancreas specifictranscription factor, 1a), and LGR5 (leucine rich repeat containing Gprotein coupled receptor 5). The protein presence of all TFs is depictedin separate single channel images. FIG. 3A (bottom) demonstrates that98.7±0.25 percent of cells were co-present for both CDX2 and SOX9 atS3D5. Strong induction of SOX9 gene expression to levels seen in Caco-2cells (FIG. 3B), and protein presence as assessed by immunofluorescence(IF)-analysis were observed (FIG. 3C). 69.4±14.2 percent of cells wereco-positive for both CDX2 and PDX1 (FIG. 3D—bottom). PDX1 geneexpression was induced at low levels, when compared to pancreas-biasedS4D3 cells (See, e.g., US2014/0242693) (FIG. 3E), and low to absentprotein levels was reflected in the IF-analysis (FIG. 3F). Anteriorendoderm TF SOX2 was not observed in S3D5 cells, as 1.45±0.15 of S3D5cells exhibited SOX2 and CDX2 co-presence (FIG. 3G—bottom; FIG. 3I), andgene expression was below levels seen in hESC and Caco-2 cells (FIG.3H). The gene expression of KLF5, essential for proper development ofintestinal mid-/hindgut endoderm, was upregulated at S3D5 (FIG. 3J).Protein co-presence of KLF5 within CDX2-positive cells at S3D5 wasobserved (FIG. 3K). ALB gene expression (FIG. 3L), and protein presence(FIG. 3M) was not observed in S3D5 cells. The gene expression ofpancreas lineage allocating TF, PTF1A, was not induced in S3D5 cells,unlike pancreas-biased S4D3 cells (FIG. 3N). The homeobox gene, HOXC5,present in the embryonic midgut endoderm was induced in S3D5 cells (FIG.3O). FIG. 3P demonstrates that LGR5, a marker of embryonic intestinalendoderm beginning at mid-gestation in the mouse, was not induced inS3D5 cells. FIG. 3Q demonstrates that HOXA13, a marker of the intestinalhindgut endoderm, was not induced in S3D5 cells (FIG. 3P). Geneexpression was assessed by qPCR.

FIGS. 4A-4B characterize the proliferative profile of differentiatingS3D5 cells. FIG. 4A depicts Caco-2 cells, where most of CDX2-proteinpositive cells were in active cell cycle (as indicated by theco-expression with KI67 protein) (left), and the proliferative index ofthe H1-hESC-derived cells during Stage 3 that decreased over time(S3D2—middle; S3D5—right). CDX2 (top row) and KI67 (bottom row) proteinlevels are depicted as single channel images. The percentageKI67-protein positive cells of total S3D5 cells (total cells are >90%CDX2-positive), assessed by FACS, was 16.8±3.12, in contrast withpercentage seen at S1D3 (97.3±1.3), and in Caco-2 cells (99.2±0.2) (FIG.4B).

FIGS. 5A-5C demonstrate use of BMP2 as an alternative to BMP4, duringStage 3 to achieve a monolayer of intestinal midgut endoderm cells withCDX2 and FOXA2 protein co-presence. FIG. 5A summarizes thedifferentiation method, including the medium components, growth factorsand small molecules that were added to each stage, and stage-specificmarkers of the differentiating intestinal midgut endoderm cells (FOXA2,CDX2, KLF5, SOX9, and PDX1^(LO)). Compared to the neutral pH noted atS2D2 (7.35±0.04), cells were exposed to slightly acidic conditions inBLAR medium, during the entirety of stage 3 (pH; S3D1, 6.92; S3D2, 7.01;S3D5, 7.22) (FIG. 5B), and as a result of lower sodium bicarbonatelevels in BLAR medium. FIG. 5C depicts representative phase-contrastimages of a S3D5 monolayer (left), and Caco-2 cells (right); a uniformmorphology at S3D5 was observed.

FIGS. 6A-6U demonstrate generation of proper intestinal midgut endodermcells in monolayer, each comprising of CDX2, FOXA2, KLF5, SOX9,PDX1^(LO) and HOXC5 on transcript and protein levels. For IF-images, allTF protein levels are depicted as single channel images. FIG. 6A(bottom) shows that 94 percent of S3D5 cells were co-present for bothCDX2 and FOXA2 protein, similar to or greater than the percentage seenin Caco-2 cells (86.0±6.67). Gene expression analysis shows CDX2 induced(FIG. 6B), and FOXA2 maintained (FIG. 6C) during Stage 3. FIG. 6D showsCDX2 protein levels and complete CDX2/FOXA2 protein co-presence inducedat S3D5, reaching similar levels as seen in Caco-2 cells (FIG. 2D-iv).FIG. 6E (bottom) demonstrates that 99.8 percent of cells were co-presentfor both CDX2 and SOX9 at S3D5. Strong induction of SOX9 gene expressionto levels seen in Caco-2 cells (FIG. 6F), and protein presence asassessed by IF-analysis were observed (FIG. 6G). 45.5 percent of cellswere co-positive for both CDX2 and PDX1 (FIG. 6H—bottom). PDX1 geneexpression was induced at low levels compared to pancreas-biased S4D3cells (FIG. 6I); low to absent protein levels was reflected in theIF-analysis (FIG. 6J). Anterior endoderm TF SOX2 was not observed inS3D5 cells, as 0.8 percent of S3D5 cells exhibited SOX2 and CDX2co-presence (FIG. 6K—bottom; FIG. 6M), and gene expression was belowlevels seen in hESC and Caco-2 cells (FIG. 6L). Gene expression of KLF5,essential for proper development of intestinal mid-/hindgut endoderm,was upregulated at S3D5 (FIG. 6N). Protein co-presence of KLFS withinCDX2-positive cells was observed at S3D5 (FIG. 6O). ALB gene expression(FIG. 6P), and protein presence (FIG. 6Q) was not observed in S3D5cells. Gene expression of pancreas lineage allocating TF, PTF1A, was notinduced in S3D5 cells, unlike pancreas-biased S4D3 cells (FIG. 6R). Thehomeobox gene HOXC5, present in the embryonic intestinal midgutendoderm, was induced in S3D5 cells (FIG. 6S). FIG. 6T demonstrates thatLGR5, a marker of embryonic intestinal endoderm beginning atmid-gestation, was not induced in S3D5 cells. FIG. 6U demonstrates thatHOXA13, a marker of the intestinal hindgut endoderm, was not induced inS3D5 cells (FIG. 6U).

FIG. 7 characterizes the proliferative profile of differentiating S3D5cells. Compared to Caco-2 cells, where most of CDX2-protein positivecells were in active cell cycle (as indicated by the co-expression withKI67 protein) (left), the proliferative index of the H1-hESC-derivedcells during Stage 3 was lower (S3D5—right). CDX2 (top row) and KI67(bottom row) protein levels are depicted as single channel images. Thepercentage KI67-protein positive cells of total S3D5 cells (total cellsare >90% CDX2-positive), assessed by FACS, was 14.1 percent, in contrastwith percentage seen at S1D3 (97.3±1.3), and in Caco-2 cells (99.2±0.2).

FIGS. 8A-8F demonstrate the induction of a heterogeneous population ofCDX2⁺ cells. FIG. 8A is a summary of the differentiation methods,including medium components, growth factors and small molecules added toeach stage, and key stage-specific markers of the differentiatingintestinal mid-/hindgut endoderm cells (HAND1). FIG. 8B showsphase-contrast images of H1-hESC cells (top row, left), post-Stage 1cells conditioned two days with 500 ng/ml FGF4 and 3 μM Chiron99021 (toprow, middle), post-Stage 1 cells conditioned two days with 500 ng/mlFGF4 and 500 ng/ml WNT3A (top row, right), a S3D5 monolayer conditionedby RA/BMP4 (bottom row, left), and a S3D5 monolayer conditioned byRA/BMP2 (bottom row, right). The induction of gene expression after twodays of conditioning is shown for CDX2 at low levels (FIG. 8C), ismaintained for the endoderm marker FOXA2 (FIG. 8D), and is induced forthe mesoderm/mesenchyme marker HAND1 (FIG. 8F). KLF5 was not induced(FIG. 8E).

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that this invention is not limited to particularmethods, reagents, compounds, compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting.

The present invention pertains to the generation of intestinal midgutendoderm cells. The cells were generated using a specific culturingsequence. Accordingly, the present invention provides an in vitro cellculture for differentiating cells derived from pluripotent stem cellsinto cells expressing markers characteristic of the intestinal midgutendoderm cell lineage, such as expression of CDX2 and FOXA2. Theinvention further provides a method for obtaining and maintaining suchcells in a monolayer via an in vitro cell culture. In certainembodiments, the invention is based on the discovery that the inclusionof retinoic acid and BMP4 or BMP2 or analogues thereof, act to induceCDX2 and maintain FOXA2 protein expression in differentiating cells tofacilitate differentiation towards intestinal midgut endoderm cells.CDX2 is not expressed at the protein level at definitive endoderm(Stage 1) or primitive gut tube (Stage 2). Accordingly, the presentinvention provides methods of differentiating pluripotent stem cells togenerate intestinal midgut endoderm cells that express CDX2 and FOXA2.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

Stem cells are undifferentiated cells defined by the ability of a singlecell both to self-renew, and to differentiate to produce progeny cells,including self-renewing progenitors, non-renewing progenitors, andterminally differentiated cells. Stem cells are also characterized bytheir ability to differentiate in vitro into functional cells of variouscell lineages from multiple germ layers (endoderm, mesoderm andectoderm), as well as to give rise to tissues of multiple germ layersfollowing transplantation, and to contribute substantially to most, ifnot all, tissues following injection into blastocysts.

Stem cells are classified according to their developmental potential as:(1) totipotent; (2) pluripotent; (3) multipotent; (4) oligopotent; and(5) unipotent. Totipotent cells are able to give rise to all embryonicand extraembryonic cell types. Pluripotent cells are able to give riseto all embryonic cell types. Multipotent cells include those able togive rise to a subset of cell lineages, but all within a particulartissue, organ, or physiological system (for example, hematopoietic stemcells (HSC) can produce progeny that include HSC (self-renewal), bloodcell-restricted oligopotent progenitors, and all cell types and elements(e.g., platelets) that are normal components of the blood). Cells thatare oligopotent can give rise to a more restricted subset of celllineages than multipotent stem cells; and cells that are unipotent areable to give rise to a single cell lineage (e.g., spermatogenic stemcells).

Stem cells are also categorized on the basis of the source from whichthey may be obtained. An adult stem cell is generally a multipotentundifferentiated cell found in tissue comprising multiple differentiatedcell types. The adult stem cell can renew itself. Under normalcircumstances, it can also differentiate to yield the specialized celltypes of the tissue from which it originated, and possibly other tissuetypes. Induced pluripotent stem cells (iPS cells) are adult cells thatare converted into pluripotent stem cells. (Takahashi et al., Cell,2006; 126(4):663-676; Takahashi et al., Cell, 2007; 131:1-12). Anembryonic stem cell is a pluripotent cell from the inner cell mass of ablastocyst-stage embryo. A fetal stem cell is one that originates fromfetal tissues or membranes.

Embryonic tissue is typically defined as tissue originating from theembryo (which in humans refers to the period from fertilization to aboutsix weeks of development). Fetal tissue refers to tissue originatingfrom the fetus, which in humans refers to the period from about sixweeks of development to parturition. Extraembryonic tissue is tissueassociated with, but not originating from, the embryo or fetus.Extraembryonic tissues include extraembryonic membranes (chorion,amnion, yolk sac and allantois), umbilical cord and placenta (whichitself forms from the chorion and the maternal decidua basalis).

Differentiation is the process by which an unspecialized (“uncommitted”)or less specialized cell acquires the features of a specialized cell,such as an intestinal cell or pancreatic cell, for example. Adifferentiated cell is one that has taken on a more specialized(“committed”) position within the lineage of a cell. The term committed,when applied to the process of differentiation, refers to a cell thathas proceeded in the differentiation pathway to a point where, undernormal circumstances, it will continue to differentiate into a specificcell type or subset of cell types, and cannot, under normalcircumstances, differentiate into a different cell type or revert to aless differentiated cell type. De-differentiation refers to the processby which a cell reverts to a less specialized (or committed) positionwithin the lineage of a cell. As used herein, the lineage of a celldefines the heredity of the cell, i.e. which cells it came from and whatcells it can give rise to. The lineage of a cell places the cell withina hereditary scheme of development and differentiation.

In a broad sense, a progenitor cell is a cell that has the capacity tocreate progeny that are more differentiated than itself, and yet retainsthe capacity to replenish the pool of progenitors. By that definition,stem cells themselves are also progenitor cells, as are the moreimmediate precursors to terminally differentiated cells. In a narrowersense, a progenitor cell is often defined as a cell that is intermediatein the differentiation pathway, i.e., it arises from a stem cell and isintermediate in the production of a mature cell type or subset of celltypes. This type of progenitor cell is generally not able to self-renew.Accordingly, if this type of cell is referred to herein, it will bereferred to as a non-renewing progenitor cell or as an intermediateprogenitor or precursor cell.

“Markers”, as used herein, are nucleic acid or polypeptide moleculesthat are differentially expressed in a cell of interest. In thiscontext, differential expression means an increased level for a positivemarker and a decreased level for a negative marker as compared to anundifferentiated cell. The detectable level of the marker nucleic acidor polypeptide is sufficiently higher or lower in the cells of interestcompared to other cells, such that the cell of interest can beidentified and distinguished from other cells using any of a variety ofmethods known in the art.

As used herein, a cell is “positive for” a specific marker or “positive”when the specific marker is sufficiently detected in the cell.Similarly, the cell is “negative for” a specific marker, or “negative”when the specific marker is not sufficiently detected in the cell. Inparticular, positive by fluorescence-activated flow cytometry (FACS) isusually greater than 2%, whereas the negative threshold by FACS isusually less than 1%.

As used herein, positive by real-time PCR (RT-PCR) had less than 28cycles (Cts), and using Taqman® Low Density Array (TLDA) had less than33 Cts; whereas negative by Open Array® is more than 28.5 cycles andnegative by TLDA is more than 33.5 Cts.

To differentiate pluripotent stem cells into functional intestinalmidgut endoderm cells in static in vitro cell culture, thedifferentiation process is often viewed as progressing throughconsecutive stages. Here, the differentiation process to intestinalmidgut endoderm occurs through three stages In this step-wiseprogression, “Stage 1” refers to the first step in the differentiationprocess, the differentiation of pluripotent stem cells into cellsexpressing markers characteristic of definitive endoderm cells(hereinafter referred to alternatively as “Stage 1 cells”). “Stage 2”refers to the second step, the differentiation of cells expressingmarkers characteristic of definitive endoderm cells into cellsexpressing markers characteristic of primitive gut tube cells(hereinafter referred to alternatively as “Stage 2 cells”). “Stage 3”refers to the third step, the differentiation of cells expressingmarkers characteristic of gut tube cells into cells expressing markerscharacteristic of intestinal midgut endoderm cells (hereinafter referredto alternatively as “Stage 3 cells”).

However, it should be noted that not all cells in a particularpopulation progress through these stages at the same rate. Consequently,it is not uncommon in in vitro cell cultures to detect the presence ofcells that have progressed less, or more, down the differentiationpathway than the majority of cells present in the population,particularly at the later differentiation stages. For purposes ofillustrating the present invention, characteristics of the various celltypes associated with the above-identified stages are described herein.

“Definitive endoderm cells,” as used herein, refers to cells which bearthe characteristics of cells arising from the epiblast duringgastrulation and which form the gastrointestinal tract and itsderivatives. Definitive endoderm cells express at least one of thefollowing markers: FOXA2 (also known as hepatocyte nuclear factor 3-β(“HNF3β”)), GATA4, SOX17, CXCR4, Brachyury, Cerberus, OTX2, goosecoid,C-Kit, CD99, and MIXL1. Markers characteristic of the definitiveendoderm cells include CXCR4, FOXA2 and SOX17. Thus, definitive endodermcells may be characterized by their expression of CXCR4, FOXA2 andSOX17. In addition, depending on the length of time cells are allowed toremain in Stage 1, an increase in HNF4α may be observed.

“Primitive gut tube cells,” as used herein, refers to endoderm cellsderived from definitive endoderm that can give rise to all endodermalorgans, such as lungs, liver, pancreas, stomach, and intestine.Primitive gut tube cells may be characterized by their substantiallyincreased expression of HNF4α over that expressed by definitive endodermcells.

“Foregut endoderm cells,” as used herein, refers to endoderm cells thatgive rise to the esophagus, lungs, stomach, liver, pancreas, gallbladder, and the most anterior portion of the duodenum. Foregut endodermcells may be characterized by their expression of SOX2, PDX1, ALB, SOX17and FOXA2, among others.

“Intestinal midgut endoderm cell,” as used herein, refers to endodermcells that give rise to small intestine. Intestinal midgut endodermcells may be characterized by their expression of CDX2, FOXA2, and lowexpression of PDX1 (PDX1^(LO)). The expression of certain HOX genes candistinguish between midgut and hindgut endoderm. For example, HOXC5 ispreferentially expressed in midgut endoderm cells.

“Hindgut endodermal cell” as used herein, refers to endoderm cells thatgive rise to large intestine. Hindgut endoderm cells may becharacterized by their expression of CDX2, FOXA2, HOXA13 and HOXD13.

“Mesenchyme cell,” as used herein, refers to mesoderm cells that giverise to connective tissues, such as bones, cartilage, lymphatic, andcirculatory systems. Expression of HAND1 and FOXF1 define mesenchymecells.

The term “patient” or “subject” or “host” refers to animals, includingmammals, preferably humans, who are treated with compositions orpharmaceutical compositions, or in accordance with the methods describedherein.

The term “effective amount” or equivalents thereof refers to an amountof an agent or compound including but not limited to a growth factor,which is sufficient to promote and differentiate human pluripotent stemcells to a differentiated cell population, for example, to a definitiveendoderm, foregut endoderm, intestinal midgut endoderm, hindgutendoderm, pancreatic endoderm and the like.

The terms “administering” and “administration” are used interchangeablyherein and mean the cells may be implanted, injected, transplanted orotherwise administered directly or indirectly to the site of treatment.When cells are administered in semi-solid or solid devices, implantationis a suitable means of delivery, particularly surgical implantation intoa precise location in the body, such as into subcutaneous space,omentum, liver, kidney (kidney capsule). Liquid or fluid pharmaceuticalcompositions may be administered to a more general location.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a cell”includes a combination of two or more cells, and the like.

As used herein, the term “about” when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of between ±20% and ±0.1%, preferably ±20% or ±10%,more preferably ±5%, even more preferably ±1%, and still more preferably±0.5%, ±0.1%. 0.05% or 0.01% of the specified value, as such variationsare appropriate to perform the disclosed methods.

The following abbreviations may appear throughout the specification andclaims:

-   ABCG2—ATP-Binding Cassette, Sub-Family G, Member 2;-   ALB—albumin;-   BMP—bone morphogenic protein;-   CDX2—caudal type homeobox 2;-   CXCR4—C-X-C chemokine receptor type 4;-   FAF-BSA—Fatty-acid Free Bovine Serum Albumin-   FGF—fibroblast growth factor;-   FOXA2—Forkhead Box A2;-   GATA4—GATA binding protein 4;-   GDF—growth differentiation factor;-   GIP—glucose-dependent insulinotropic polypeptide;-   GLP-1—glucagon-like peptide 1;-   GSK3B—Glycogen synthase kinase 3 beta;-   HAND1—heart and neural crest derivatives expressed 1;-   HOX—homeobox;-   hTERT—human telomerase reverse transcriptase;-   KLF—Kruppel-like factor;-   LGR5—leucine rich repeat containing G protein coupled receptor 5;-   MIXL1—Mix Paired-Like Homeobox-1;-   OCT4—octamer-binding transcription factor 4;-   OTX2—orthodenticle homeobox 2;-   PDX1—pancreatic and duodenal homeobox 1;-   PTF1A—pancreas specific transcription factor, 1a;-   SOX—sex determining region Y (SRY)-box;-   TRA1-60—T cell receptor alpha-1-60;-   UTF1—Undifferentiated embryonic cell transcription factor 1;-   WNT3A—wingless-type MMTV integration site family, member 3A; and-   ZFP42—zinc finger protein 42.

DETAILED DESCRIPTION

Pluripotent stem cells have the potential to differentiate into cells ofall three germinal layers: endoderm, mesoderm, and ectoderm tissues.Exemplary types of pluripotent stem cells that may be used includeestablished lines of pluripotent cells, including pre-embryonic tissue(such as, for example, a blastocyst), embryonic tissue, or fetal tissuetaken any time during gestation, typically but not necessarily, beforeapproximately 10 to 12 weeks gestation. Non-limiting examples areestablished lines of human embryonic stem cells or human embryonic germcells, such as, for example the human embryonic stem cell lines H1, H7,and H9 (WiCell Research Institute, Madison, Wis., USA). Cells taken froma pluripotent stem cell population already cultured in the absence offeeder cells are also suitable. iPS, or reprogrammed pluripotent cells,derived from adult somatic cells using forced expression of a number ofpluripotent related transcription factors, such as OCT4, NANOG, SOX2,KLF4, and ZFP42 (Annu Rev Genomics Hum Genet 2011, 12:165-185; see alsoiPS, Cell, 126(4): 663-676) may also be used. The human embryonic stemcells used in the methods of the invention may also be prepared asdescribed by Thomson et al. (U.S. Pat. No. 5,843,780; Science, 1998,282:1145-1147; Curr Top Dev Biol 1998, 38:133-165; Proc Natl Acad SciU.S.A. 1995, 92:7844-7848). Mutant human embryonic stem cell lines, suchas, BG01v (BresaGen, Athens, Ga.), or cells derived from adult humansomatic cells, such as, cells disclosed in Takahashi et al., Cell 131:1-12 (2007) may also be used. In certain embodiments, pluripotent stemcells suitable for use in the present invention may be derived accordingto the methods described in: Li et al. (Cell Stem Cell 4: 16-19, 2009);Maherali et al. (Cell Stem Cell 1: 55-70, 2007); Stadtfeld et al. (CellStem Cell 2: 230-240); Nakagawa et al. (Nature Biotechnol 26: 101-106,2008); Takahashi et al. (Cell 131: 861-872, 2007); and U.S. Patent App.Pub. No. 2011/0104805. In certain embodiments, the pluripotent stemcells may be of non-embryonic origins. All of these references, patents,and patent applications are herein incorporated by reference in theirentirety, in particular, as they pertain to the isolation, culture,expansion and differentiation of pluripotent cells.

Pluripotent stem cells differentiate through various stages each ofwhich may be characterized by the presence or absence of particularmarkers. Differentiation of the cells into these stages is achieved bythe specific culturing conditions, including the presence or lack ofcertain factors added to the culture media. In general, thisdifferentiation may involve differentiation of pluripotent stem cellsinto definitive endoderm cells, referred to herein as Stage 1. Thesedefinitive endoderm cells may then be further differentiated intoprimitive gut tube cells, referred to herein as Stage 2. Primitive guttube cells in turn may then be differentiated into intestinal midgutendoderm cells, referred to herein as Stage 3.

Differentiation of Pluripotent Stem Cells into Cells Expressing MarkersCharacteristic of Intestinal Midgut Endoderm Cells

Characteristics of pluripotent stem cells are well known to thoseskilled in the art, and additional characteristics of pluripotent stemcells continue to be identified. Pluripotent stem cell markers include,for example, the expression of one or more of the following: ABCG2;Cripto; FOXD3; CONNEXIN43; CONNEXIN45; OCT4; SOX2; NANOG; hTERT; UTF1;ZFP42; SSEA-3; SSEA-4; TRA-1-60; and TRA-1-81.

Exemplary pluripotent stem cells include the human embryonic stem cellline H1 (NIH code: WA01), the human embryonic stem cell line H9 (NIHcode: WA09), the human embryonic stem cell line H7 (NIH code: WA07), andthe human embryonic stem cell line SA002 (Cellartis, Sweden). Alsosuitable are cells that express at least one of the following markerscharacteristic of pluripotent cells: ABCG2, Cripto, CD9, FOXD3,CONNEXIN43, CONNEXIN45, OCT4, SOX2, NANOG, hTERT, UTF1, ZFP42, SSEA-3,SSEA-4, TRA-1-60, and TRA-1-81.

Also suitable for use in the present invention is a cell that expressesat least one of the markers characteristic of the definitive endodermlineage. In one embodiment of the invention, a cell expressing markerscharacteristic of the definitive endoderm lineage is a primitive streakprecursor cell. In an alternate embodiment, a cell expressing markerscharacteristic of the definitive endoderm lineage is a mesendoderm cell.In an alternate embodiment, a cell expressing markers characteristic ofthe definitive endoderm lineage is a definitive endoderm cell.

Also suitable for use in the present invention is a cell that expressesat least one of the markers characteristic of the intestinal midgutendoderm lineage. In one embodiment of the present invention, a cellexpressing markers characteristic of the intestinal endoderm lineage isan intestinal midgut endoderm cell in which the cell expresses FOXA2 andCDX2. In some embodiments, the cell does not express SOX2, ALB, PTF1A,HOXA13 or LGR5. In embodiments, a cell expressing markers characteristicof the intestinal endoderm lineage is an intestinal midgut endoderm cellin which the cell expresses each of FOXA2, CDX2, SOX9, PDX1, KLF5 andHOXC5. In embodiments, a cell expressing markers characteristic of theintestinal endoderm lineage is an intestinal midgut endoderm cell inwhich the cell does not express any of SOX2, ALB, PTF1A, HOXA13 andLGR5.

The invention provides for staged, directed differentiation ofpluripotent stem cells toward intestinal midgut endoderm cells usingcell culture conditions and media. In embodiments of the invention, toarrive at a cell expressing markers characteristic of intestinal midgutendoderm cell, a protocol starting with pluripotent stem cells, such asembryonic stem cells and induced pluripotent cells, is employed. Thisprotocol includes the following stages.

-   -   Stage 1: Pluripotent stem cells, such as embryonic stem cells        obtained for cell culture lines, are treated with appropriate        factors to induce differentiation into cells expressing markers        characteristic of definitive endoderm cells.    -   Stage 2: Cells resulting from Stage 1 are treated with        appropriate factors to induce further differentiation into cells        expressing markers characteristic of primitive gut tube cells.    -   Stage 3: Cells resulting from Stage 2 are treated with        appropriate factors to induce further differentiation into cells        expressing markers characteristic of intestinal midgut endoderm        cells.

Methods for assessing expression of protein and nucleic acid markers incultured or isolated cells are standard in the art. These methodsinclude RT-PCR, Northern blots, in situ hybridization (see, e.g.,Current Protocols in Molecular Biology (Ausubel et al., eds. 2001supplement)), and immunoassays (such as immunohistochemical analysis ofsectioned material), Western blotting, and for markers that areaccessible in intact cells, FACS (see, e.g., Harlow and Lane, UsingAntibodies: A Laboratory Manual, New York: Cold Spring Harbor LaboratoryPress (1998)). The efficiency of differentiation may be determined byexposing a treated cell population to an agent (such as an antibody)that specifically recognizes a protein marker expressed by cellsexpressing markers characteristic of the cell type of interest.

1. Differentiation of Pluripotent Stem Cells into Cells ExpressingMarkers Characteristic of Definitive Endoderm Cells

Pluripotent stem cells may be differentiated into cells expressingmarkers characteristic of definitive endoderm cells by any suitablemethod known in the art, or by any method proposed in this invention. Inone embodiment of the invention, pluripotent stem cells are treated witha medium, such as MCDB-131 medium (Life Technologies, Carlsbad, Calif.)supplemented with factors including GDF8 and a GSK3β inhibitor (such asthe cyclic aniline-pyridinotriazine compounds disclosed in U.S. PatentApp. Pub. No. 2010/0015711; incorporated herein by reference in itsentirety) to induce differentiation into cells expressing markerscharacteristic of definitive endoderm cells. There is a broad range ofGSK3β inhibitors, such as staurosporine, and the preferred GSK3βinhibitor (14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one,referred to herein as “MCX Compound”. Treatment may involve contactingpluripotent stem cells with a medium supplemented with about 10 ng/ml to1000 ng/ml, preferably 50 ng/ml to about 150 ng/ml, alternatively about75 ng/ml to about 125 ng/ml, alternatively about 100 ng/ml of GDF8. Thetreatment may also involve contacting the cells with about 0.1 to 10 μM,preferably about 0.1 to 5 μM, alternatively about 0.5 to about 2.5 μM,preferable about 1.5 μM or about 1.0 μM of MCX compound. Othercomponents of the medium may include: Sodium bicarbonate at about 2.7g/1000 ml to 3.6 g/1000 ml, preferably 2.7 g/1000 ml; FAF-BSA at about0.1% to 2.0%, preferably about 0.5%; GlutaMAX™ (Life TechnologiesCorporation, Carlsbad, Calif.) at 1:100 dilution (“1× concentration”);and D-Glucose at a concentration range of about 2 mM to 20 mM,preferably 4.5 mM to obtain a concentration of 10 mM D-Glucose.

The pluripotent cells may be cultured for approximately two to fivedays, preferably about three to four days, to facilitate theirdifferentiation into definitive endoderm cells. In one embodiment, thepluripotent cells are cultured in the presence of an effective amount ofTGFβ signaling molecule and/or GSK3β inhibitor, for example, aneffective amount of GDF8 and MCX compound for one day, followed byculturing in the presence of GDF8 and a lower concentration of MCXcompound for one day, followed by culturing in the presence of GDF8 forone day in the absence of the MCX compound. In particular, the cells maybe cultured in the presence of GDF8 and about 1.5 μM of MCX compound forone day, followed by culturing in the presence of GDF8 and about 0.1 μMMCX compound for one day, followed by culturing in the presence of GDF8for one day in the absence of the MCX compound. In an alternateembodiment, the cells may be cultured in the presence of GDF8 and about1.5 μM of MCX compound for one day, followed by culturing in thepresence of GDF8 and about 0.1 μM MCX compound for one day.

Generation of cells expressing markers characteristic of definitiveendoderm cells may be determined by testing for the presence of themarkers before and after following a particular protocol. Pluripotentstem cells typically do not express such markers. Thus, differentiationof pluripotent cells can be detected when the cells begin to expressmarkers characteristic of definitive endoderm cells, such as CXCR4,FOXA2 and SOX17. In the embodiments, cells expressing markerscharacteristic of definitive endoderm cells are definitive endodermcells.

2. Differentiation of Cells Expressing Markers Characteristic ofDefinitive Endoderm Cells into Cells Expressing Markers Characteristicof Primitive Gut Tube Cells

The cells expressing markers characteristic of definitive endoderm cellsmay be further differentiated into cells expressing markerscharacteristic of primitive gut tube cells. In one embodiment, theformation of cells expressing markers characteristic of primitive guttube cells includes culturing cells expressing markers characteristic ofdefinitive endoderm cells with a medium, such as MCDB-131, containingFGF7 to differentiate these cells. For example, the culture medium mayinclude from about 10 ng/ml to 100 ng/ml, preferably about 25 ng/ml toabout 75 ng/ml, alternatively from about 30 ng/ml to about 60 ng/ml,alternatively about 50 ng/ml of FGF7. The cells may be cultured underthese conditions for about two to three days, preferably about two days.

In another embodiment, differentiation into cells expressing markerscharacteristic of primitive gut tube cells includes culturing cellsexpressing markers characteristic of definitive endoderm cells with FGF7and ascorbic acid (vitamin C). The culture medium, such as MCDB-131, mayinclude from about 0.1 mM to about 1.0 mM ascorbic acid, preferablyabout 0.1 mM to about 1.0 mM, alternatively from about 0.2 mM to about0.4 mM, alternatively about 0.25 mM of ascorbic acid. The culture mediummay also include from about 10 ng/ml to 100 ng/ml, preferably about 10ng/ml to about 50 ng/ml, alternatively from about 15 ng/ml to about 30ng/ml, alternatively about 50 ng/ml or about 25 ng/ml of FGF7. Forexample, the culture medium may include about 0.25 mM ascorbic acid andabout 50 ng/ml FGF7. Other components of the medium may include: sodiumbicarbonate at about 2.7 g/1000 ml to 3.6 g/1000 ml, preferably 2.7g/1000 ml; FAF-BSA at about 0.1% to 2.0%, preferably about 0.5%;GlutaMAX™ at 1:100 dilution (“1× concentration”); and D-Glucose at aconcentration range of about 2 mM to 20 mM, preferably 4.5 mM to obtaina concentration of 10 mM D-Glucose. In one embodiment, cells expressingmarkers characteristic of definitive endoderm cells are treated for 2days with FGF7 and ascorbic acid. Differentiation of definitive endodermcells can be detected when the cells begin to express markerscharacteristic of primitive gut tube cells, such as expression of FOXA2and increased expression of HNF4α. In the embodiments, cells expressingmarkers characteristic of primitive gut tube cells are primitive guttube cells.

3. Differentiation of Cells Expressing Markers Characteristic ofPrimitive Gut Tube Cells into Cells Expressing Markers Characteristic ofIntestinal Midgut Endoderm Cells

Cells expressing markers characteristic of primitive gut tube cells maybe further differentiated into cells expressing markers characteristicof intestinal midgut endoderm cells. In one embodiment, primitive guttube cells are further differentiated into intestinal midgut endodermcells by culturing the primitive gut tube cells in a culture medium,such as BLAR medium (Life Technologies, Corporation, Carlsbad, Calif.),supplemented with retinoic acid and a BMP4 or BMP2. In a preferredembodiment, the medium is supplemented with from about 0.5 μM to about 5μM of retinoic acid, preferably about 1 μM, and from about 10 ng/ml toabout 100 ng/ml BMP4 or BMP2, preferably about 50 ng/ml of BMP4 or BMP2.Other supplements to the medium may include: FAF-BSA at about 0.1% to2.0%, preferably about 0.5%; GlutaMAX™; and D-Glucose at a concentrationrange of about 2 mM to 20 mM, preferably 4.5 mM to obtain aconcentration of 10 mM D-Glucose. In one embodiment, cells expressingmarkers characteristic of primitive gut cells are treated for 3 to 5days, preferably for 5 days with BMP4 or BMP2 and retinoic acid. The pHof the culture can range from 6.8 to 7.2 during the 5-day Stage 3conditioning period (compared to normal pH at S2D2 being 7.3 or more).

The invention relates to a method of producing a population ofintestinal midgut endoderm cells by culturing human pluripotent stemcells in selected culture media for generating intestinal midgutendoderm cells. In embodiments, the method induces differentiation ofhuman pluripotent stem cell to intestinal midgut endoderm cells in astaged process. In embodiments, a population of intestinal midgutendoderm cells is produced. In some embodiments, a population ofsubstantially intestinal midgut endoderm cells is produced. In theembodiments, the intestinal midgut endoderm cells form and maintain amonolayer on planar culture. In embodiments, the intestinal midgutendoderm cells are stable as a monolayer in culture. Cells stable as amonolayer, or remain stable as a monolayer, herein refers to a monolayerof cells that do not form spheroids in culture.

In embodiments, greater than 50% of the differentiated cells expressmarkers characteristic of intestinal midgut endoderm. In embodiments,greater than 60%, greater than 70%, greater than 80%, greater than 90%or greater than 95% of the differentiated cells express markerscharacteristic of intestinal midgut endoderm. In embodiments,differentiated cells express markers characteristic of intestinal midgutendoderm are intestinal midgut endoderm cells. In embodiments, theintestinal midgut endoderm cells express CDX2 and FOXA2 as determined byFACS analysis and qPCR. In some embodiments, intestinal midgut endodermcells express transcription factors selected from SOX9, PDX1, KLF5 andHOXC5 as determined by IF analysis and qPCR. In embodiments, theintestinal midgut endoderm cells do not express transcription factorsselected from SOX2, ALB, PTF1A as determined by IF analysis and qPCR,and HOXA13 and LGR5 as determined by qPCR.

A further embodiment of the invention is a method of producingintestinal midgut endoderm cells comprising inducing differentiation ofdefinitive endoderm cells in culture to primitive gut tube cells. Inembodiments, the definitive endoderm cells are cultured in culture mediacontaining ascorbic acid and FGF7. In further embodiments, the primitivegut tube cells are cultured in culture media containing retinoic acidand BMP2 or BMP4. The primitive gut tube cells are differentiated tointestinal midgut endoderm cells. In some embodiments, primitive guttube cells are differentiated to intestinal midgut endoderm cells inacidic conditions (acidic culture media). In particular embodiments,acidic conditions is culture in BLAR media. The pH of the acidic culturecan range from 6.8 to 7.2 during the 5-day differentiation conditioningperiod from primitive gut tube cells to intestinal midgut endoderm cells(compared to normal pH at S2D2 being 7.3 or more). In the embodiments,the intestinal midgut endoderm cells form and maintain a monolayer inculture.

In each of the embodiments discussed herein, human pluripotent stemcells are human hESC or iPS cells. In each of the embodiments above andherein, the intestinal midgut endoderm cells express CDX2 and FOXA2 asdetermined by FACS analysis and qPCR. In all embodiments, the intestinalmidgut endoderm cells express transcription factors selected from SOX9,PDX1, KLF5 and HOXC5 as determined by IF analysis and qPCR. In theembodiments above and herein, the intestinal midgut endoderm cells donot express transcription factors selected from SOX2, ALB, PTF1A asdetermined by IF analysis and qPCR, and HOXA13 and LGR5 as determined byqPCR. In the embodiments above and herein, the intestinal midgutendoderm cells express CDX2, FOXA2, SOX9, PDX1, KLF5 and HOXC5 by IFanalysis and qPCR. In each of the embodiments, the intestinal midgutendoderm cells do not express SOX2, ALB and PTF1A as determined by IFanalysis and qPCR, and HOXA13 and LGR5 as determined by qPCR.

As a result of the differentiation protocol described above and herein,using specific culture components and culture conditions, in particularacidic culture condition, such as culture in BLAR medium, a culture ofcells expressing markers for intestinal midgut endoderm cells isgenerated; the cells lack expression of HAND1 as determined by qPCR, amarker of mesoderm/mesenchymal lineage. Varying the differentiationprotocol to induce pluripotent stem cells to midgut/hindgut endodermlineage, such as inducing stem cells at definitive endoderm stage 1rather than primitive gut tube cell stage 2, results in a mixedpopulation of endoderm-mesenchyme CDX2+ mid-/hindgut cells that expressHAND1 as determined by qPCR.

In certain embodiments, the population of intestinal midgut endodermcells is substantially intestinal midgut endoderm cells. In someembodiments, the population of intestinal midgut endoderm cellscomprises greater than 70% intestinal midgut endoderm cells, preferablygreater than 80%, greater than 90%, and greater than 95% of intestinalmidgut endoderm cells. In some embodiments, the population of intestinalmidgut endoderm cells comprises less than 20% mesenchymal cells,preferably less than 15%, more preferably less than 10%, less than 5%,less than 2%, less than 1%, less than 0.5%. In embodiments, intestinalmidgut endoderm cells lack expression of HAND1.

Use of Differentiated Intestinal Midgut Endoderm Cells

In another embodiment of the invention, the differentiated intestinalmidgut endoderm cells may be used for treating a patient suffering fromor at risk of developing diabetes alone or in combination withdifferentiated or mature endocrine cells, for example, enteroendocrinecells. In such embodiments, differentiated intestinal midgut endodermcells, or mixtures thereof, may be administered to a patient havingdiabetes, for example Type 1 or Type 2 diabetes. In embodiments,intestinal midgut endoderm cells differentiate and mature toenteroendocrine cells. In embodiments, intestinal midgut endoderm cellsdifferentiate and mature to enteroendocrine cells, and theenteroendocrine cells express or secrete incretin type hormones. Inembodiments, incretin hormones include GLP1 and GIP. Administration ofthe cells may be via implantation or injection in the body, inparticular implantation into subcutaneous space, omentum, liver, kidney,etc.

In some embodiments of the invention described above, differentiation ofintestinal midgut endoderm cells is induced in vitro. In otherembodiments, intestinal midgut endoderm cells further differentiate andmature in vivo. Another embodiment relates to the intestinal midgutendoderm cells further differentiating into enteroendocrine cells invivo or in a mixture with enteroendocrine cells in vivo. Suchenteroendocrine cells express or secrete incretin hormones. Inembodiments, the enteroendocrine cell-secreted incretin hormones includeGLP1 and GIP.

In a further embodiment, intestinal midgut endoderm cells serve asstarting material for the identification of small molecules that promoteat high efficiency the in vitro differentiation of intestinal midgutendoderm cell type into, first, enteroendocrine precursors, and then toincretin expressing or secreting enteroendocrine cells.

Cells and cell populations and mixtures such as those described hereinmay be micro- or macro-encapsulated and subsequently transplanted into amammalian host. Encapsulated cells or cells alone may be transplanted(administered) subcutaneously or anywhere else in the body whereby thecells may be vascularized and differentiate and mature in vivo.

EXAMPLES

The invention can be further understood in view of the followingnon-limiting examples.

Example 1 Method of Producing an Intestinal Midgut Endoderm CellPopulation with CDX2 and FOXA2 Co-Presence/Co-Expression

The following example describes a directed-based method to generateintestinal midgut endoderm cells from human embryonic stem cell(“hESC”). “Intestinal midgut endoderm” refers to a corresponding in vivoor in situ cell type which is CDX2-positive and FOXA2-positive endodermcells present at about embryonic day 8.5 (“E8.5”) during mousedevelopment, or at about the 3-4 week time point during human embryonicdevelopment.

Materials and Methods

Cell culture: Cells of the human embryonic stem cell line H1 (“H1-hESC”)(WA01 cells, WiCell Research Institute, Madison, Wis.) cultured with EZ8media (Cat #A1516901 Gibco, Thermo Fisher Scientific) at passage 28 wereseeded as single cells at 0.094×10⁶ cells/cm² on MATRIGEL™, at a 1:30dilution, (Corning Incorporated, Corning, N.Y., Catalog #356231) coateddishes in a media of Dulbecco's Modified Eagle's Medium Nutrient mixtureF-12 (“DMEM-F12”) (Life Technologies Corporation, Carlsbad, Calif.,Catalog No. 11330-032), with the following:

GlutaMAX ™ 1:100 dilution (Life Technologies Corporation, Carlsbad, (“1×concentration”) California, Catalog No. 35050-079) Ascorbic acid 0.25 mM(Sigma Aldrich Co. LLC, St. Louis, Missouri, Catalog No. A4544) FGF2 100 ng/ml (R & D Systems Inc., Minneapolis, Minnesota, Catalog No.233-FB-025) Transforming Growth Factor beta (“TGFβ”)   1 ng/ml (R & DSystems Inc., Minneapolis, Minnesota, Catalog No. 240-B-002)insulin-transferrin-selenium-ethanolamine 1:200 dilution (“ITS-X”) (LifeTechnologies, Carlsbad, California, Catalog No. 51500056) at a 1:100dilution FAF-BSA 2% (Proliant, Inc., Boone, Idaho, Catalog No. 68700)Insulin-like Growth Factor-1 (“IGF-1”)   20 ng/ml (R & D Systems Inc.,Minneapolis, Minnesota, Catalog No. 291-G1-200) Rock Inhibitor Y-27632(“Y-compound”)   10 μM (Sigma Aldrich Co. LLC, St. Louis, Missouri,Catalog No. Y-0503)

About forty-eight hours post-seeding, the cultures were washed inincomplete PBS (phosphate buffered saline without magnesium or calcium)(Life Technologies, Carlsbad, Calif., Catalog No. 14190). Rock InhibitorY-27632 (Y compound) was used only for the first 24 hours of culture.

Differentiation: The cultures were differentiated using the followingprotocol. During Stages 1 through 3 of the protocol, cultures weremaintained on planar adherent cultures. Others, however, have describeddifferentiation using suspension culture including US US2014/0242693,which is incorporated by reference in its entirety; the protocoldescribed herein can be modified and performed in suspension, whichprovides for scalability of manufacturing. The following nomenclature,S#D#, specifies exact time during Stages 1 through 3. For example, S1D3is stage 1 day 3. Briefly, each stage defines differentiation towardsdefinitive endoderm (stage 1), primitive gut tube (stage 2), andintestinal midgut endoderm (stage 3).

a. Stage 1 (3 days): Cells were cultured for one day in the followingStage 1 media:

MCDB-131 medium (Life Technologies, Carlsbad, California, Catalog No.ME120219L2) Sodium bicarbonate  2.7 g/1000 ml (Sigma-Aldrich Co. LLC,St. Louis, Missouri, Catalog No. 5761) FAF-BSA 0.5% GlutaMAX ™ 1:100dilution (“1× concentration”) D-Glucose  4.5 mM to obtain a(Sigma-Aldrich Co. LLC, St. Louis, concentration of 10 mM Missouri,Catalog No. G8769) D-Glucose GDF8  100 ng/ml (Peprotech, Rocky Hill, NewJersey, Catalog No. 120-00) 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-  1.5 μMheptaazatetracyclo [19.3.1.1~2,6~.1~8,12~]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen- 16-one (“MCX compound”).

-   -   Cells were then cultured for an additional day in the following        media:

MCDB-131 medium Sodium bicarbonate  2.7 g/1000 ml FAF-BSA 0.5%GlutaMAX ™ 1× concentration D-Glucose  4.5 mM to obtain a concentrationof 10 mM D-Glucose GDF8  100 ng/ml MCX compound  0.1 μM

-   -   Cells were then cultured for an additional day in the same media        as day 2 above but without MCX compound.

b. Stage 2 (2 days): Cells were treated for two days with the followingmedium:

MCDB-131 medium Sodium bicarbonate  2.7 g/1000 ml FAF-BSA 0.5%GlutaMAX ™ 1× concentration D-Glucose  4.5 mM to obtain a concentrationof 10 mM D-Glucose Ascorbic Acid 0.25 mM FGF7   50 ng/ml (R & D Systems,Inc., Minneapolis, Minnesota, Catalog No. 251-K

c. Stage 3 (5 days): Cells were treated for five day with BLAR 001custom medium:

BLAR medium (custom manufactured by Life Technologies, Corporation,Carlsbad, California, Catalog No. ME120123L2, components listed on TableI) FAF-BSA 0.5% GlutaMAX ™ 1× concentration D-Glucose 4.5 mM to obtain aconcentration of 10 mM D-Glucose Retinoic Acid   1 μM (Sigma Aldrich,St. Louis, Missouri, Catalog No. R2625) BMP4 compound  50 ng/ml (R & DSystems, Inc., Minneapolis, Minnesota, Catalog No. 314-BP OR BMP2compound (R & D Systems, Inc., Minneapolis, Minnesota, Catalog No.355-BM),

Either BMP4 or BMP2 can be used during Stage 3 in this method to achieveintestinal midgut endoderm cells in monolayer with CDX2 and FOXA2protein co-presence, as illustrated in FIGS. 1A-4B (BMP4-based), andFIGS. 5A-7 (BMP2-based).

TABLE I List of components of BLAR medium. Concentration (mM) AminoAcids Glycine 3.00E−02 Alanine 3.00E−02 Arginine 3.00E−01 Asparagine1.00E−01 Aspartic Acid 1.00E−01 Cysteine 1.99E−01 Glutamic acid 3.00E−02Histidine 1.10E−01 Isoleucine 1.00E−02 Leucine 9.00E−02 Lysinehydrochloride 1.50E−01 Methionine 3.00E−02 Phenylalanine 3.00E−02Proline 1.00E−01 Serine 1.00E−01 Threonine 3.00E−02 Tryptophan 2.00E−03Tyrosine disodium 1.00E−02 Valine 3.00E−02 Vitamins Biotin 3.00E−05Choline chloride 5.00E−03 D-Calcium pantothenate 1.50E−03 Folinic AcidCalcium salt 2.30E−03 Niacinamide 4.90E−03 Pyridoxine hydrochloride9.70E−04 Riboflavin 1.00E−05 Thiamine hydrochloride 3.00E−03 Vitamin B123.70E−06 i-Inositol 2.80E−03 Salts/Minerals Calcium Chloride(CaCl₂—2H₂O) 3.00E−01 Cupric sulfate (CuSO₄—5H₂O) 4.80E−06 Ferricsulfate (FeSO₄—7H₂O) 1.00E−03 Magnesium Sulfate (MgSO₄—7H₂O) 4.10E−01Potassium Chloride (KCl) 3.80E+00 Sodium Bicarbonate (NaHCO₃) 1.40E+01Sodium Chloride (NaCl) 1.10E+02 Sodium Phosphate dibasic 5.00E−01(Na₂HPO₄—7H₂O) Zinc Sulfate (ZnSO4—H2O) 1.00E−04 Other Adenine 1.00E−03D-Glucose (Dextrose) 5.00E+00 Lipoic Acid 1.20E−05 Phenol Red 1.00E−02Sodium Pyruvate 1.00E+00 Thymidine 9.80E−05

Quantification of differentiated cells: For quantification of proteinpresence co-localization, S3D5 cells were harvested and analyzed byFACS. FACS staining was conducted as described in Nature Biotechnology,2014 (32) 11, 1121-1133, incorporated herein by reference in itsentirety, and using the antibodies listed in Table II. In brief,differentiated cells were incubated in TrypLE™ Express (LifeTechnologies, Carlsbad, Calif., Catalog No. 12604) for 5-10 minutes at37° C., released into a single-cell suspension, after which they werewashed twice with a staining buffer of PBS containing 0.2% BSA (BDBiosciences, San Jose, Calif., Catalog No. 554657). Intracellularantibody staining was accomplished by utilizing the LIVE/DEAD VioletFluorescent reactive dye (Life Technologies, Carlsbad, Calif., CatalogNo. L34955) at 4° C. for 30 minutes followed by a single wash in coldPBS. Fixing of cells was in 300 μl of Cytofix/Cytoperm Buffer (BDBiosciences, San Jose, Calif., Catalog No. 554723) followed by twowashes in Perm/Wash Buffer (BD Biosciences, San Jose, Calif., CatalogNo. 554722). Cells were then incubated with the appropriate antibodiesat 4° C. for 30 minutes (for unconjugated antibodies) or 1 hour (forconjugated antibodies), and then washed twice prior to analysis on theBD FACS Canto II using BD FACS Diva Software with at least 30,000 eventsbeing acquired. Non-viable cells were excluded during FACS analysis, andgating was determined by using isotype antibodies (“IgG”). IgG FACS datais shown as the top panel for each FACS experiment presented. Antibodieswere tested for specificity using positive controls, such as Caco-2cells, or negative controls, such as S1D3 definitive endoderm (“DE”)cells.

TABLE II List of antibodies used for FACS analysis. Source/CatalogAntigen Species Number Dilution PE IgG1, κ Isotype control Mouse BD Cat#555749 1:5 Alexa Fluor 647 IgG1, κ Mouse BD Cat# 557732 Neat Isotypecontrol PE anti-Human Mouse BD Cat# 561589 Neat FOXA2 PE Anti-human CDX2Mouse BD Cat# 563428 Neat Alexa Fluor 647 anti- Mouse BD Cat# 560395Neat CDX2 Alexa Fluor 647 Mouse BD Cat# 561126 Neat anti-KI67 PEanti-PDX1 Mouse BD Cat# 562161 Neat PE anti-SOX2 Mouse BD Cat# 560291Neat Anti-Mouse IgG(H + L) Goat Life technology 1:4000 SecondaryAntibody, Cat# A21235 Alexa Fluor 647 conjugate F(ab′)2 anti-rabbit GoatLife technology Neat IgG(H + L) Secondary Cat# A10542 Antibody, RPEconjugate Anti-SOX9 Rabbit Millipore Cat# 1:10 AB5535 Anti-CDX2 MouseBioGenex Cat# 1:10 [CDX2-88] MU392A-UC

For quantification of protein co-localization at various stages, Caco-2,S2D2, S3D2 and S3D5 cells were harvested as a monolayer and analyzed byimmunofluorescence (“IF”). Note that the morphology seen in IF imageswas caused by the method of cell scraping from the monolayer of adherentcultures. H1-hESC-derived cells were prepared and stained as describedin Nature Biotechnology, 2014 (32) 11, 1121-1133, and using theantibodies listed in Table III. For cryosectioning, cells were rinsedwith PBS followed by overnight fixation in 4% PFA (Sigma Aldrich, St.Louis, Mo., Catalog No. 158127) at 4° C. Following fixation, 4% PFA wasremoved, cells rinsed twice with PBS, and incubated overnight at 4° C.in 30% sucrose solution (Amresco, Solon, Ohio, Catalog No. 0335). Thesamples were cryopreserved in OCT solution (Sakura Finetek USA Inc.,Torrance, Calif., Catalog No. 4583), and 5 μm sections placed onSuperfrost plus slides (VWR International, LLC, Radnor, Pa., Catalog No.48311-703). For IF-staining, primary antibodies were added atappropriate dilutions overnight at 4° C., while secondary antibodieswere added for 30 min at room temperature followed by rinsing with PBSand adding Vectastain mounting reagent with DAPI (Vector LaboratoriesInc., Burlingame, California, Catalog No. H-1200). The sections werevisualized using a Nikon Ti fluorescence microscope (Nikon Instruments,Inc., Melville, N.Y.).

TABLE III List of antibodies used for IF analysis. Antigen SpeciesSource Dilution CDX2 Mouse BioGenex 1:50 (Catalog No. MU392A-UC) FOXA2Rabbit Seven Hills 1:500 (Catalog No. WRAB 1200) SOX9 Rabbit MilliporeEMD 1:100 (Catalog No. AB5535) PDX1 Goat R & D Systems 1:33 (Catalog No.AF2419) SOX2 Goat Sigma 1:50 (Catalog No. sc-17320) KLF5 Rabbit Abcam1:150 (Catalog No. ab24331) (Tyramide amplification- Perkin Elmer,Waltham, MA, NEL744001KT) ALB Rabbit Sigma 1:250 (Catalog No. A0433)KI67 Rabbit Abcam 1:100 (Catalog No. ab16667) Donkey anti-mouse IgGMouse Life Technologies 1:100 (H + L) Secondary (Catalog No. A21202)antibody, Alexa Fluor 488 Donkey anti-rabbit IgG Rabbit LifeTechnologies 1:200 (H + L) Secondary (Catalog No. A10040) Antibody,Alexa Fluor 546 Donkey anti-goat IgG Goat Life Technologies 1:50-1:100(H + L) Secondary (Catalog No. A11056) Antibody, Alexa Fluor 546 Goatanti-rabbit IgG, Rabbit Perkin Elmer, Waltham, MA 1:250 HRP-Labeled(Catalog No. NEF812001)

For quantification of gene expression at various stages, Caco-2,H1-hESC, S1D3, S2D2, S3D2 and S3D5 cells were harvested, as described inNature Biotechnology, 2014 (32) 11, 1121-1133. Briefly, gene expressionwas assessed in cells using custom Taqman® Arrays (Applied Biosystems,Foster City, Calif.); Open Array® (OA) was used for CDX2, FOXA2, SOX2,SOX9, PDX1, ALB, PTF1A, and Taqman® Low Density Array (TLDA) was usedfor KLF5, HOXC5, and LGR5, with housekeeping gene GAPDH used for bothtests. Data were analyzed using Sequence Detection Software (AppliedBiosystems, Foster City, Calif.), and normalized using GAPDH as ahousekeeping gene to undifferentiated H1-hESC using the ΔΔCt method.Primer details are outlined in Table IV.

TABLE IV List of RT-qPCR primers. Gene Assay ID  1 ALB Hs00609411_m1  2CDX2 Hs00230919_m1  3 FOXA2 Hs00232764_m1  4 GAPDH Hs99999905_m1  5 PDX1Hs00236830_m1  6 PTF1A Hs00603586_g1  7 SOX2 Hs01053049_s1  8 SOX9Hs00165814_m1  9 HOXC5 Hs00232747_m1 10 KLF5 Hs00156145_m1 11 LGR5Hs00969422_m1 12 HOXA13 Hs00426284_m1 13 HAND1 Hs00231848_m1

Results

A summary of a differentiation method, including the important mediumcomponents, growth factors and small molecules that were added to eachstage, and key stage-specific markers of the differentiating intestinalmidgut endoderm cells (FOXA2; CDX2; KLF5; SOX9; PDX1^(LO)) is depictedin FIG. 1A. As compared to the neutral pH noted for S2D2 (7.35±0.04),cells were exposed to slightly acidic conditions in the BLAR medium,during the entirety of stage 3 (pH; S3D1, 6.98±0.05; S3D2, 7.02±0.04;S3D5, 7.18±0.03) (FIG. 1B), and as a result of lower sodium bicarbonatelevels in BLAR medium. The pH of the culture can range from about 6.8 to7.2 during the five days of stage 3. FIG. 1C depicts representativephase-contrast images of a S3D5 monolayer (left), and human epithelialcolon adenocarcinoma cell line (“Caco-2”) (right), which was used as abenchmark for characterization of differentiation. A uniform morphologyat S3D5 was observed. Characterization of cell number using theNucleocounter® NC-100 (Chemometec, Alleroed, Denmark , Catalog No.900-004) shows that one hESC differentiated into 4.56±2.60 S3D5 hindgutendoderm cells (FIG. 1D).

The differentiation method, utilizing BMP4, efficiently generates andmaintains intestinal midgut endoderm cells in monolayer, each comprisingboth CDX2 and FOXA2 on transcript and protein levels. FIG. 2A (bottom)shows that 90.0±5.85 percent of S3D5 cells were co-present for both CDX2and FOXA2 protein, similar to percentage observed in Caco-2 cells(86.0±6.67). Conversely, definitive endoderm (DE—S1D3) cells were devoidof CDX2 and FOXA2 co-presence (2.3±1.2). Gene expression analysis showsCDX2 induced (FIG. 2B), and FOXA2 maintained (FIG. 2C) during Stage 3.FIG. 2D shows that the induction of CDX2 protein levels and CDX2/FOXA2protein co-presence after the establishment of the FOXA2-positiveprimitive gut endoderm stage, S2D2 (FIG. 2D-i), progressively increasedthrough S3D2 (FIG. 2D-ii), and at S3D5 (FIG. 2D-iii) reached similarlevels as seen in Caco-2 cells (FIG. 2D-iv). CDX2 protein is depicted onthe bottom row, and FOXA2 protein is depicted on the top row.

Transcript and protein levels of additional TFs are found at S3D5, whichconstitute robust intestinal midgut endoderm induction. FIGS. 3A-3P showthat proper intestinal midgut endoderm was achieved. In addition to CDX2and FOXA2 co-presence, S3D5 cells also exhibited co-presence of SOX9,PDX1, KLF5, HOXC5, but did not express SOX2, ALB, PTF1A, and LGR5. Theprotein presence of all TFs is depicted in separate single channelimages. FIG. 3A (bottom) shows that 98.7±0.25 percent of cells wereco-present for both CDX2 and SOX9 at S3D5. Strong induction of SOX9 geneexpression was comparable to levels observed in Caco-2 cells (FIG. 3B),and protein presence as assessed by IF-analysis were observed (FIG. 3C).69.4±14.2 percent of cells were co-positive for both CDX2 and PDX1 (FIG.3D—bottom). PDX1 gene expression was induced at low levels, as comparedto pancreas-biased S4D3 cells (See, e.g., US2014/0242693) (FIG. 3E), andthis was reflected low to absent protein levels in the IF-analysis (FIG.3F).

S3D5 cells did not express the anterior endoderm TF SOX2, and only1.45±0.15 of S3D5 cells exhibited SOX2 and CDX2 co-presence (FIG.3G—bottom; 3I), and gene expression was below levels seen in hESC andCaco-2 cells (FIG. 3H). Gene expression of KLF5, essential for properdevelopment of hindgut endoderm, was upregulated at S3D5 (FIG. 3J).Protein co-presence of KLF5 within CDX2-positive cells at S3D5 wasobserved (FIG. 3K). ALB gene expression (FIG. 3L), and protein presence(FIG. 3M) was not observed in S3D5 cells. Similarly, the gene expressionof PTF1A, a pancreas lineage marker, was not induced in S3D5 cells, ascompared to pancreas-biased S4D3 cells (FIG. 3N). The homeobox gene,HOXC5, present in the embryonic intestinal midgut endoderm was stronglyinduced in S3D5 cells (FIG. 3O). FIG. 3P shows that LGR5, a marker ofembryonic intestinal endoderm beginning at mid-gestation in the mouse,was not induced in S3D5 cells (FIG. 3P). FIG. 3Q shows that HOXA13, amarker of the intestinal hindgut endoderm, was not induced in S3D5 cells(FIG. 3P).

FIGS. 4A-4B illustrate the proliferative profile of differentiating S3D5cells. FIG. 4A depicts Caco-2 cells, where most of CDX2-protein positivecells were in active cell cycle (as indicated by the co-expression withKI67 protein) (left), and the proliferative index of the H1-hESC-derivedcells during Stage 3 that decreased over time (S3D2—middle; S3D5—right).CDX2 (top row) and KI67 (bottom row) protein levels are depicted assingle channel images. The percentage KI67-protein positive cells oftotal S3D5 cells (total cells are >90% CDX2-positive), assessed by FACS,was 16.8±3.12, in contrast with percentage seen at S1D3 (97.3±1.3), andin Caco-2 cells (99.2±0.2) (FIG. 4B).

BMP2 can be used as an alternative to BMP4, during Stage 3 in thismethod to produce a monolayer of intestinal midgut endoderm cells withCDX2 and FOXA2 protein co-presence. FIG. 5A depicts a summary of adifferentiation method, including the medium components, growth factorsand small molecules that were added to each stage, and keystage-specific markers of the differentiating intestinal midgut endodermcells (FOXA2, CDX2, KLF5, SOX9, and PDX1^(LO)). Compared to the neutralpH noted at S2D2 (7.35±0.04), cells were exposed to slightly acidicconditions in BLAR medium, during the entirety of stage 3 (pH; S3D1,6.92; S3D2, 7.01; S3D5, 7.22) (FIG. 5B), and as a result of lower sodiumbicarbonate levels in BLAR acidic medium. FIG. 5C depicts representativephase-contrast images of a S3D5 monolayer (left), and Caco-2 cells(right). A uniform morphology at S3D5 was observed.

The differentiation method generates and maintains proper intestinalmidgut endoderm cells in monolayer, each comprising of CDX2, FOXA2,KLF5, SOX9, PDX1^(LO) and HOXC5 on transcript and protein levels. All TFprotein levels are depicted as single channel IF images. FIG. 6A(bottom) shows that 94 percent of S3D5 cells were co-present for bothCDX2 and FOXA2 protein, similar to percentage seen in Caco-2 cells(86.0±6.67). Gene expression analysis shows that CDX2 was induced (FIG.6B), and FOXA2 maintained (FIG. 6C) during Stage 3. FIG. 6D shows thatCDX2 protein levels and complete CDX2/FOXA2 protein co-presence wereinduced at S3D5 (FIG. 6D), which is comparable to levels observed inCaco-2 cells (FIG. 2D-iv). FIG. 6E (bottom) shows that 99.8 percent ofcells were co-present for both CDX2 and SOX9 at S3D5. Strong inductionof SOX9 gene expression was observed similar to levels in Caco-2 cells(FIG. 6F), and protein presence as assessed by IF-analysis was observed(FIG. 6G). 45.5 percent of cells were co-positive for both CDX2 and PDX1(FIG. 6H—bottom). PDX1 gene expression was induced at low levels, whencompared to pancreas-biased S4D3 cells (FIG. 6I), and low to absentprotein levels was reflected in the IF-analysis (FIG. 6J). Anteriorendoderm TF SOX2 was not observed in S3D5 cells, as 0.8 percent of S3D5cells exhibited SOX2 and CDX2 co-presence (FIG. 6K—bottom; 6M), and geneexpression was below levels seen in hESC and Caco-2 cells (FIG. 6L). Thegene expression of KLF5, an essential marker for demonstrating properdevelopment of hindgut endoderm, was strongly upregulated at S3D5 (FIG.6N). Protein co-presence of KLF5 within CDX2-positive cells at S3D5 wasobserved (FIG. 6O). ALB gene expression (FIG. 6P), and protein presence(FIG. 6Q) was not observed in S3D5 cells. The gene expression ofpancreas lineage allocating TF, PTF1A, was not induced in S3D5 cells, ascompared to pancreas-biased S4D3 cells (FIG. 6R). The homeobox geneHOXC5, present in the embryonic intestinal midgut endoderm, was stronglyinduced in S3D5 cells (FIG. 6S). FIG. 6T demonstrates that LGR5, amarker of embryonic intestinal endoderm beginning at mid-gestation, wasnot induced in S3D5 cells. FIG. 6U demonstrates that HOXA13, a marker ofthe intestinal hindgut endoderm, was not induced in S3D5 cells (FIG.6U).

FIG. 7 characterizes the proliferative profile of differentiating S3D5cells, showing Caco-2 cells, where most of CDX2-protein positive cellswere in active cell cycle (as indicated by the co-expression with KI67protein) (left), compared to the proliferative index of theH1-hESC-derived cells during Stage 3 that was lower (S3D5—right). CDX2(top row) and KI67 (bottom row) protein levels are depicted as singlechannel images. The percentage KI67-protein positive cells of total S3D5cells (total cells are >90% CDX2-positive), as assessed by FACS, was14.1 percent, in contrast with percentage seen at S1D3 (97.3±1.3),compared to Caco-2 cells (99.2±0.2) (FIG. 7; 4B).

EXAMPLE 2 Intestinal Culturing Starting from the Definitive Endoderm,and Using FGF4 and WNT-Agonists, Generates an Endoderm-MesenchymeMixture of CDX2+ Mid-/Hindgut Cells

This example demonstrates the endoderm-mesenchyme-mixed quality of theCDX2+ mid-/hindgut cells generated from intestinal culturing beginningat the definitive endoderm stage using FGF4 and WNT-agonists (Spence etal., Nature, 2011; 470:105-109; Watson et al. Nature Med, 2014;11:1310-1314). To examine the induction to midgut/hindgut endoderm cellsdescribed in Spence et al., infra, hESCs were differentiated using theprotocol below. Note that the differentiation conditions outlined inthis Example differ from Example 1 by the following: (i) intestinalcondition starting point begins at the definitive endoderm stage; (ii)different growth factors and small molecules are used than RA and BMP4or BMP2; and (iii) acidic culture conditions are not used.

Materials and Methods

Cell culture: H1-hESC cells were cultured and maintained as described inExample 1.

Differentiation: The cultures were differentiated using the followingprotocol.

-   Stage 1—Mimic (3 days): Cells were cultured for one day in the    following Stage 1 medium:

RMPI 1640 medium (Thermo Fisher Scientific, Catalog No. 11875)Penicillin-Streptomycin 1× concentration (1:100 dilution (Thermo FisherScientific, Catalog No. from stock concentration) 15140122) L-Glutamine 2 mM (Thermo Fisher Scientific, Catalog No. 25030081) Activin A 100ng/ml (R & D Systems, Inc., Minneapolis, Minnesota, Catalog No. 338-AC)

Cells were then cultured for an additional day in the following media:

RMPI 1640 medium Penicillin-Streptomycin 1× concentration (1:100dilution from stock concentration) L-Glutamine  2 mM Activin A 100 ng/mlDefined Fetal Bovine Serum (FBS) 0.2% (Hyclone, Catalog No. SH30070.02)

Cells were then cultured for an additional day in the following media:

RMPI 1640 medium Penicillin-Streptomycin 1× concentration (1:100dilution from stock concentration) L-Glutamine  2 mM Activin A 100 ng/mlDefined FBS 2.0%

Post-Stage 1 (2 days): For example, pS1d1 is post-Stage 1 day 1 andpS1d2 is post-Stage 1 day 2. Cells were cultured for two days in thefollowing Post-Stage 1 medium:

RMPI 1640 medium Penicillin-Streptomycin 1× concentration (1:100dilution from stock concentration) L-Glutamine  2 mM FGF4 500 ng/ml (R &D Systems, Inc., Minneapolis, Minnesota, Catalog No. 235-F4) DefinedFetal Bovine Serum (FBS) 2.0% WNT3A 500 ng/ml (R & D Systems, Inc.,Minneapolis, Minnesota, Catalog No. 5036-WN) OR Chiron99201  3 μM(Stemgent, Catalog No. 0400041)

Quantification: Phase contrast imaging and quantification of geneexpression followed the procedures in Example 1.

Results

A summary of the differentiation methods, including medium components,growth factors and small molecules added to each stage, and signature orkey stage-specific markers of the differentiating intestinalmid-/hindgut endoderm cells (HAND1) is depicted in FIG. 8A. Intestinalconditioning (post-Stage 1), started at the definitive endoderm stage,with 500 ng/ml FGF4, and either 3 μM Chiron99021 (Watson et al.), or 500ng/ml Wnt3A (Spence et al.). The term “Stage 1-mimic” refers to thedefinitive endoderm differentiation protocol in this example thatdiffers from the “S1D3-Original” that refers to Stage 1 conditioningdescribed in Example 1. FIG. 8B shows phase-contrast images of H1-hESCcells (top row, left), post-Stage 1 cells conditioned two days with 500ng/ml FGF4 and 3 μM Chiron99021 (top row, middle), post-Stage 1 cellsconditioned two days with 500 ng/ml FGF4 and 500 ng/ml Wnt3A (top row,right), along with a S3D5 monolayer conditioned by RA/BMP4 (bottom row,left), and a S3D5 monolayer conditioned by RA/BMP2 (bottom row, right)(from Example 1).

Induction of CDX2 gene expression was achieved after two days ofconditioning, but at much lower levels compared to RA/BMP2 or RA/BMP4S3D5 (FIG. 8C). However, the gene expression of the endoderm markerFOXA2 was maintained (FIG. 8D), and mesoderm/mesenchyme marker HAND1 wasstrongly induced (FIG. 8F). Also, KLF5 was not induced at the two daytime point unlike by RA/BMP4 and RA/BMP2 conditioning (FIG. 8E). Asconclusively shown in FIG. 8F, this gene expression pattern isreflective of the heterogeneous cell population seen in Watson et al.and Spence et al. containing not only a CDX2⁺FOXA2⁺ endodermalpopulation, but also a significant mesenchymal CDX2⁺ cell population. Incontrast, RA/BMP4 or RA/BMP2 post-Stage 2 (primitive gut tube cell)conditioning did not induce the mesoderm/mesenchyme marker HAND1; onlyendodermal CDX2⁺FOXA2⁺ population was induced.

In describing the present invention and its various embodiments,specific terminology is employed for the sake of clarity. However, theinvention is not intended to be limited to the specific terminology soselected. A person skilled in the relevant art will recognize that otherequivalent components can be employed and other methods developedwithout departing from the broad concepts of the current invention. Allreferences cited anywhere in this specification are incorporated byreference as if each had been individually incorporated.

1. A population of substantially intestinal midgut endoderm cells,wherein the population of cells is encapsulated.
 2. The population ofsubstantially intestinal midgut endoderm cells of claim 1, wherein thepopulation of cells is micro-encapsulated.
 3. The population ofsubstantially intestinal midgut endoderm cells of claim 1, wherein thepopulation cells is macro-encapsulated.
 4. The population ofsubstantially intestinal midgut endoderm cells of claim 1, wherein theintestinal midgut endoderm cells express CDX2 and FOXA2.
 5. Thepopulation of substantially intestinal midgut endoderm cells of claim 1,wherein the intestinal midgut endoderm cells express transcriptionfactors selected from the group consisting of SOX9, PDX1, KLF5 andHOXC5.
 6. The population of substantially intestinal midgut endodermcells of claim 1, wherein the intestinal midgut endoderm cells do notexpress transcription factors selected from the group consisting ofSOX2, ALB, PTF1A, HOXA13 and LGR5.
 7. The population of substantiallyintestinal midgut endoderm cells of claim 1, wherein the intestinalmidgut endoderm cells do not express HAND1.
 8. The population ofsubstantially intestinal midgut endoderm cells of claim 1, wherein thepopulation comprises greater than 70% intestinal midgut endoderm cells.9. The population of substantially intestinal midgut endoderm cells ofclaim 8, wherein the population comprises greater than 80% intestinalmidgut endoderm cells.
 10. The population of substantially intestinalmidgut endoderm cells of claim 8, wherein the population comprisesgreater than 90% intestinal midgut endoderm cells.
 11. The population ofsubstantially intestinal midgut endoderm cells of claim 1, wherein thepopulation comprises less than 20% mesenchymal cells.
 12. The populationof substantially intestinal midgut endoderm cells of claim 11, whereinthe population comprises less than 10% mesenchymal cells.
 13. Thepopulation of substantially intestinal midgut endoderm cells of claim11, wherein the population comprises less than 1% mesenchymal cells. 14.The population of substantially intestinal midgut endoderm cells ofclaim 1, wherein the population does not include mesenchymal cells.